Exploring voltage sensing in protein-translocating channel SecYEG.

Abstract

The conserved protein translocation channel SecYEG facilitates protein transport across as well as integration into the membrane. To maintain ion gradients across the membrane, essential for many cellular processes, it is of utmost importance that the Sec channel remains impermeable to these ions despite its bulky substrates. It has been shown via electrophysiological experiments that physiological levels of transmembrane voltage allow the channel to stay impermeable. Yet, the substrate-bound SecYEG becomes leaky once the absolute value of the transmembrane voltage drops below a certain threshold [1,2]. We proposed that the dipole moment of helix 2b is responsible for voltage sensing and that other complex parts must also contribute [3]. Molecular dynamics simulations suggested the involvement of the 5th helix dipole. Replacing S188 on its cytoplasmatic terminus allowed reverting its electrostatic contribution. Here, we measured the kinetics of voltage-gated closings of the thus mutated bacterial SecYEG translocon, reconstituted into planar bilayers. The translocon openings were triggered by the binding of ribosome nascent chain complexes harboring the voltage sensor domain of the voltage-gated potassium channel KvAP. S188E exhibited an increased voltage sensitivity, whereas S188R partially de-sensitized the translocon. Our results confirm the earlier hypothesis, in which the dipole moment of multiple TM-helices serves as SecYEG's voltage sensor. Support of the Austrian Science Fund (FWF), grant P34584 to PP, is gratefully acknowledged.